Stabilization of live viral vaccines by hexahydric alcohols



United States Patent ice STABILIZATION OF LIVE VIRAL VACCINES BYHEXAHYDRIC ALCOHOLS Stewart Aiston, Congers, and Irvin S. Danielson,Pearl River, N.Y., assignors to American Cyanamid Company, New York,N.Y., a corporation of Maine Application November 5, 1954 Serial No.467,213

11 Claims. (Cl. 167-78) No Drawing.

The present invention relates to vaccines and to methods of preparingthe same. More particularly, the invention is concerned with improvedvaccines containing live microorganisms selected from the groupconsisting of viruses and rickettsiae of increased stability andpossessing effective potency over longer periods of time.

Vaccines are preparations normally consisting of a V microorganism suchas bacteria, rickettsia or virus which has been treated or manipulatedto reduce or to attenuate its virulence and whichis used forpreventative inoculation to induce active immunity by the productionwithin the animalbody of antibodies against the specific microorganismused in the vaccine. preparations lose their eflicacy and potency quiterapidly, especially when exposed to room temperature for prolongedperiods of time. Storage under refrigeration Substantially all of thesesomewhat increases the life of these preparations but, even under themost desirable conditions, their properties are shortlived.Consequently, only small inventories of fresh supplies of such vaccinesmay be kept on hand in hospitals, research facilities and laboratoriesand must be replenished at all too frequent intervals. The problem isfurther complicated by the increasing number and variety of vaccinesavailable for use particularly in the case of the smaller or more remotelaboratories which cannot practically or conveniently store the manytypes required.

It is highly desirable, therefore, and a principal object of the presentinvention to provide vaccines containing live microorganisms selectedfrom the group consisting of viruses and rickettsiae of greaterstability and longer storagelife whereby larger amounts of a greatervariety of such supplies may be safely stored with a minimized lossappreciated that these alcohols may be used individually (I or inadmixture with one another, as desired.

Among the vaccines which have been found to be satisfactorily applicablewithin the concepts of the present invention may be cited the vaccinesfor diseases such as small pox, yellow fever, distemper, hog cholera,fowl pox,

Newcastle disease, pigeon pox, rabies, poliomyelitis, etc.

It is to be observed that these vaccines contain live micro organismsand further are cited primarily as illustrative of the applications ofthe invention and are not tov be construed as limitative thereof.

The amount of the hexahydric alcohol to be incor n,

rated in the vaccines for stabilizing purposes will depend on manyfactors such as the nature of the vaccine itself, its properties andcharacteristics, the form wherein it is to be stored, and the like. Forexample, in dried vaccines, amounts as low as about 0.5% or as high asabout 810% or higher, based on the dry weight of the hexahydroxyalcohol, have been found satisfactory with preferred commercial rangeslying between about 2% and about 7%. In the case of fluid vaccines,wherein drying characteristics and properties are of lesser importance,similar amounts may be used, although amounts of up to about 60% ormore, based on the dry weight of the hexahydroxy alcohol, may beadvantageously employed for increased stabilizing effects, particularlywhere aqueous solutions are involved, with preferred commercial rangeslying between about 10% and about 50%.

Any suitable standard procedure may be employed to prepare the vaccine.The microorganisms may be obtained from any suitable source, such as,for example, from a stock strain or directly from an animal or patientand propagated by Well known methods in chick embryo tissue or extraembryonic membranes, calf lymph, tissue culture, or other suitablemedia. A finely divided emulsion may then be formed and mixed or blendedwith the selected hexahydric alcohol in the desired amount. If desired,the selected polyhydric may be mixed or blended initially with the virusprior to the formation of the finely divided emulsion. The alcohol maybe employed either in the dried crystalline form or in a concentratedcommercial solution, such as Arlex or Sorbo 70%.

The invention will be further described by reference to the followingexamples setting forth in specific details several preferred embodimentsof the inventive concept as well as comparisons to vaccines presentlyused of lesser stability and of much shorter lives. It should beunderstood, however, that these examples and accompanying tables aregiven primarily for illustrative purposes and the inventive concept inits broader aspects is not to be construed as limited thereto. Allpercentages are by weight on a dry basis, unless otherwise stated.

EXAMPLE I The following data will illustrate the stabilizing effectexerted by sorbitol when added to live virus vaccines (poliomyelitis,Lansing type) which had been vacuum dried and stored at roomtemperature. In this data, the percentage chick embryo (C E.) is thepercentage suspension of homogenized infected chick embryo. The titersare expressed in LB as determined and by intra- Small poxvacc'ine (live)was used in this example and was produced from the membranes ofsmallpox-infected embryonated eggs. The infected membranes werehar-wvested, homogenized and suspended in the fluids indicated. The potencyof the preparations before and after incuba tion at 35 C. was judged bythe takes developing in rabbits when serial dilutions of the vaccinewere applied to scarified areas of skin of the rabbits.Allpreparationswere dried from the frozen state. Homogenizedinfectedchick embryo membranes suspended in: (A) distilled: water were used inobtaining the data'rof TableQlIA;

0 l (B) distilled water containing sucrose and phosphate salts in TableHE; (C) 7% sorbitol containing 5% peptone in Table HA {Distilled water.}

Percent Percent Percent Percent Percent takes takes takes vtakes takes1:1,000 1:3,000 1110,000 1130,000 l:100,000

Before incubation v(Sample 1) 97 77 3,9 12 9 After 16 days incubatlon(l) 0 0 0 0 Before incubation 98 76 30 18 6 l After 16 days incubation(2) 5 11 p 0 0 '0 Table IIB [Distilled water.]

Percent Percent Percent Percent Percent takes akes takes takes akes121,000 1:3,000 1110,000 1230,000 100,000

Before incubation ample 3) 97 77 39 12 9 After 16 days incuatlon (3) 378 D 0 0 Before incubation (Sample 4) 98 7B 30 18 6 After 16 daysineubatlon (4) 16 2 0 0 0 Table IIC [7% sorbitoL] Percent PercentPercent Percent Percent takes takes takes takes takes 1:1,000 1:3,0001:10,000 30,000 1:100,000

Before incubation am e5) l. 98 89 80 77v 64 After 30 days incubation (5)'98 80 63 50 0 Before incubation ample 98 94 75 57 9 After 30 daysincubation (6) 90 90 55 16 i 12 Table 11D [7% sorbitoL] Percent PercentPercent Percent Percent takes takes takes takes takes 1:1,000 1:3,000l:10,000 1230,000 l:100,0(l0

Before incubation am e 98 98 82 81 42 After 30 days incubation (7) 93 8044 7 15 EXAMPLE In The following preparations were made from homogenizedchick embryos which had been infected with live rabies virus (Flurystrain). A 33% suspension was prepared and sorbitol was added asindicated in Table III.

4 EXAMPLE IV Table IV Concentratlon per ml.

Titer 21 days Agent Mixtures of sorbitol and mannitol, sorbitol andinositol, and mannitol and inositol were employed in similar samples andtiters after 7 days and 21 days at 37 C. ranged from l0- -10- to 10 -10-respectively, as calculated by the Reed-Munch technique.

EXAMPLE V The following data will illustrate the stabilizing efiectexerted on fluid small pox vaccines containing various percentages ofglycerine and sorbitol. The figures given in the respective columns ofTables VA and VB take ind'ices for rabbits as obtained by the followingtests:

Each experimental sample of vaccine and a known control viruspreparation are diluted at 1:1000, 1:3000, 1:l0,000, 130,000 and1:100,000, usually in beef heart infusion broth. For each titration,five superficial scratches of about 5 cm. in length are made on theshaved flanks of a rabbit with a sharp needle. Each of the abovedilutions is then rubber vigorously into one screatch with the help ofthe blunt and of a glass tube. Inoculated rabbits are kept in individualcages for 5 days at which time the length of the scratch and the extentof the lesion are measured in millimeters and the percentage of thelesion, or take, calculated. The mean average of takes obtained with thefive dilutions of the control virus is considered as a take index of1.0. This then allows the calculation of the take indices of theexperimental vaccines tested simultancously with the control Vll'llS.

Below is an illustration for calculation of a takeindcx.

All preparations were vacuum dried from the frozen state. The potencydata was calculated as LD values obtained by injecting mice with serialdilutions of each preparation, c m- Virus mental calculated by the ReedMunch technique. Dilutions 55 I a Table 111 8 fiiif LDu Values after-1:1000 96 moon so 95 Percent sorbitol added 150,000 89 90 7 days at 14days at 1:30,!)00- 66 26 37 0. W0. 70 1:10am 26 30 Total 866 336 (I10-33 0 10- 10- :Mean Average 73. 2 67,2 2 1Q-3JE 104.0! K 10-84"- Takeindex =o.02.

Although several specific examples of the inventive concept have beendescribed, the same should not be construed as limited thereby nor tothe specific substances mentioned therein but to include various othercompounds of equivalent constitution as set forth in the claims appendedhereto. It is understood that any suitable changes, modifications andvariations may be made without departing from the spirit and scope ofthe invention.

What we claim is:

1. A biological product comprising live avirulent virus effective ineliciting the production of immunizing antibodies in animals and 0.5percent to 60 percent by weight of an alcohol of the group consisting ofsorbitol, mannitol, inositol, and dulcitol, said alcohol having astabilizing efiect on said live virus whereby the vaccine has aneifective immunizing potency over an extended period of time.

2. A biological product comprising live avirulent virus eifective ineliciting the production of immunizing antibodies against smallpox inanimals and 0.5 percent to 60 percent by weight of an alcohol of thegroup consisting of sorbitol, mannitol, inositol, and dulcitol, saidalcohol having a stabilizing efiect on said live virus whereby thevaccine has an efiective immunizing potency over an extended period oftime.

3. A biological product comprising live attenuated poliomyelitis viruseffective in eliciting the production of immunizing antibodies inanimals and 0.5 percent to 60 percent by weight of an alcohol of thegroup consisting of sorbitol, mannitol, inositol, and dulcitol, saidalcohol having a stabilizing effect on said live virus whereby thevaccine has an effective immunizing potency over an extended period oftime.

4. A biological product comprising live attenuated rabies viruseffective in eliciting the production of immunizing antibodies inanimals and 0.5 percent to 60 percent by weight of an alcohol of thegroup consisting of soribitol, mannitol, inositol, and dulcitol, saidalcohol iv. i I "TAKE INDIOES OF DIFFERENT BATOHES 0F FLUID SMALLPOXVACCINE, CHICK EMBRYO ORIGIN r.

Unlncubatad CapillaryTubes 1ncuhatedat35-37 O. I Samples I Diluent BulkCapilla lDay 2Days 3Daya 4Days 5Days 6Days 7 Days 14 21 Tubes Days Days50% Glycerin 1.11 0.90 0.83" 0.13 p 0.07 0 0.02

Do '1. 07, 1. 07 0. 74 0. 52 0.05 W130.bit l 0.72 0.54 0.29 0.07 0. 120.09

O1 0 gm 0.64 51 0.40 0.34 0.26 0.13

or 0. 94 0.85 0.00 0. 59 i gs or 37 3 0.0a 0.03 0.21 0.05 0.01 0 21 1.050.00 0.48 0.19 0.04 0 gl ffif 1.12 0. 7s 0. 56 0.30 0.0a 0 60% sorbitol.1.05 0.92 0.71 0.73 0.61 0; 02

Table VB TAKE INDIOES" 0F DIFFEgIAQIIZIQ SMALLPOX VACCINE,

Unincubatsd Capillary Tubes Incubated at 35-37 0.

Samples Dlluent Bulk Capillary 1 2 3 5 7 14 21 Tubes Day Days Days DaysDays Days Days 50% Glycerin 1.08 1.13 0. 60 0.21 0.10 0.03 Do-- 1. 31 1.29 0.72 0. 32 0. 14 0.07 Do 0. 88 0. 88 0. 54 0. 19 0. l5 0. 07 Do 1.29 1. 24 0. 71 0.20 0.11 50% Sorbito 1.08 0. 74 0. 62 0. 64 0.40 0.17 035% Sorbltoll. 01 0. 94 0. 64 0. 72 0.45 0. 21 0.01 50% sorbitol--- 1.20 1. 29 0. 92 0. 87 0.83 0. 37 0.02 Do 1. 24 1. 11 0. 81 0. 75 0. B7 0.39 0. 11

having a stabilizing effect on said live virus whereby the vaccine hasan etfective immunizing potency over an extended period of time.

5. A biological product comprising live avirulent virus eifective ineliciting the production of immunizing antibodies in animals and 0.5percent to 60 percent by weight of sorbitol, said sorbitol having astabilizing effect on said live virus whereby the vaccine has aneffective immunizing potency over an extended period of time.

6. A biological product comprising live avirulent virus effective ineliciting the production of immunizing antibodies in animals and 0.5percent to 60 percent by weight of mannitol, said mannitol having astabilizing effect on said live virus whereby the vaccine has anefiective immunizing potency over an extended period of time.

7. A biological product comprising live avirulent virus effective ineliciting the production of immunizing antibodies in animals and 0.5percent to 60 percent by weight of inositol, said inositol having astabilizing efiect on said live virus whereby the vaccine has aneffective immunizing potency over an extended period of time.

8. A method of stabilizing live avirulent virus products containing liveavirulent virus particles eficctive in eliciting the production ofimmunizing antibodies in animals, which comprises adding to saidproducts from 0.5 percent to 60 percent by weight of an alcohol of thegroup consisting of sorbitol, mannitol, inositol, and dulcitol, saidalcohol having a stabilizing effect on said live virus whereby thevaccine has an effective immunizing potency over an extended period oftime.

9. A method of stabilizing a live avirulent virus product whichcomprises adding to said product containing live avirulent virusparticles effective in eliciting the production of immunizing antibodiesagainst smallpox in animals from 0.5 percent to 60 percent by weight ofan alcohol of the group consisting of sorbitol, mannitol,

inositol, and dulcitol, said alcohol having a stabilizing elfect on saidlive virus whereby the vaccine has an effective immunizing potency overan extended period ing antibodies in animals, which comprises adding tosaid product from 0.5 rpercent to 60percen1 by weighti'szf an alcohol ofthe group consisting of sorbitol, mannitol, inositol, and dulc'ito'i,said alcohol having a stabilizing effect on said live virus-wherebythe'vacciu e has an efiec- 10 five immunizing po n y cverlan extendedperiod @of t me.

11. A method ,of stabilizing a live attenuated rabies virus productcontaining live attenuated virus particles effective in eliciting the-production of immunizing antibodies in animals, whichppmprises addingto .saidyroduct from 0.5 percent to 60 percent by weight of an alcoholof the group consisting of sorbitol, mannito'l, iiuositol, and dulcitol,said alcohol having astabilizing eiiect on said live virus :whereby the:vaccinehas an teffec tive immunizing potency over an extended period.of time.

8 "References Cited in the file of this patent .FQRE'IGN ,PAIENIS26,148/30 Australia Apr. 11, 1930 28,066 Great Britain December 1913OTHER REFERENCES Franklinetal: .Proc. Soc. iExptl. Biol. and Med., vol.95; \,-pp- 715-718 =(th u rRea e s a d M dia :fe Tissue Culture andVirus Propagation, pub. by Difco,

Nature, vol. 168, December 8, 1-951, -pp.-990 and 9 9;1 F y: A ic e inez ng and D yin nub "by that.

15 of BioL, London, June 1951,- pp. 107-115.

la s i h" y l fl owsler C0, w lm n tcn Levine: ,QQ P ati n o QultureMedia tier the (jlultivation of Microorganisms, by Williamsand 20 pp.40, @425, 875, 881 and 888.

1. A BIOLOGICAL PRODUCT COMPRISING LIVE AVIRULENT VIRUS EFFECTIVE INELICITING THE PRODUCTION OF IMMUNIZING ANTIBODIES IN ANIMALS AND 0.5PERCENT TO 60 PERCENT BY WEIGHT OF AN ALCOHOL OF THE GROUP CONSISTING OFSORBITOL, MANNITOL, INOSITOL, AND DULCITOL, SAID ALCOHOL HAVING ASTABILIZING EFFECT ON SAID LIVE VIRUS WHEREBY THE VACCINE HAS ANEFFECTIVE IMMUNIZING POTENCY OVER AN EXTENDED PERIOD OF TIME.